Development of a Systematic qPCR Array for Screening GM Soybeans

A screening method using the 35S promoter and nos terminator for genetically modified organisms (GMOs) is not sufficient to cover all GM soybean events. In this study, a real-time polymerase chain reaction (also known as quantitative polymerase chain reaction, qPCR) array targeting eight screening assays combined with a prediction system was developed for the rapid tracking of GM soybeans. Each assay’s specificity was tested and confirmed using 17 GM soybean events that have been approved in Korea. The sensitivity of each assay was determined to range from 0.01% to 0.05% using DNA mixtures with different GM ratios, and it was validated by the results of three experimenters. The applicability of this study was tested by monitoring 23 processed foods containing soybeans. It was figured out that 13 of the 23 samples included GM soybeans. The prediction system combined with screening results will be helpful to trace the absence/presence of GM soybean events. This new qPCR array and prediction system for GM soybean detection provides rapid, convenient and reliable results to users

Therapeutic Status and Available Strategies in Pancreatic Ductal Adenocarcinoma

One of the most severe and devastating cancer is pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the major pancreatic exocrine cancer with a poor prognosis and growing prevalence. It is the most deadly disease, with an overall five-year survival rate of 6% to 10%. According to various reports, it has been demonstrated that pancreatic cancer stem cells (PCSCs) are the main factor responsible for the tumor development, proliferation, resistance to anti-cancer drugs, and recurrence of tumors after surgery. PCSCs have encouraged new therapeutic methods to be explored that can specifically target cancer cells. Furthermore, stem cells, especially mesenchymal stem cells (MSCs), are known as influential anti-cancer agents as they function through anti-inflammatory, paracrine, cytokines, and chemokine′s action. The properties of MSCs, such as migration to the site of infection and host immune cell activation by its secretome, seem to control the microenvironment of the pancreatic tumor. MSCs secretome exhibits similar therapeutic advantages as a conventional cell-based therapy. Moreover, the potential for drug delivery could be enhanced by engineered MSCs to increase drug bioactivity and absorption at the tumor site. In this review, we have discussed available therapeutic strategies, treatment hurdles, and the role of different factors such as PCSCs, cysteine, GPCR, PKM2, signaling pathways, immunotherapy, and NK-based therapy in pancreatic cancer

Therapeutic Status and Available Strategies in Pancreatic Ductal Adenocarcinoma

One of the most severe and devastating cancer is pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the major pancreatic exocrine cancer with a poor prognosis and growing prevalence. It is the most deadly disease, with an overall five-year survival rate of 6% to 10%. According to various reports, it has been demonstrated that pancreatic cancer stem cells (PCSCs) are the main factor responsible for the tumor development, proliferation, resistance to anti-cancer drugs, and recurrence of tumors after surgery. PCSCs have encouraged new therapeutic methods to be explored that can specifically target cancer cells. Furthermore, stem cells, especially mesenchymal stem cells (MSCs), are known as influential anti-cancer agents as they function through anti-inflammatory, paracrine, cytokines, and chemokine′s action. The properties of MSCs, such as migration to the site of infection and host immune cell activation by its secretome, seem to control the microenvironment of the pancreatic tumor. MSCs secretome exhibits similar therapeutic advantages as a conventional cell-based therapy. Moreover, the potential for drug delivery could be enhanced by engineered MSCs to increase drug bioactivity and absorption at the tumor site. In this review, we have discussed available therapeutic strategies, treatment hurdles, and the role of different factors such as PCSCs, cysteine, GPCR, PKM2, signaling pathways, immunotherapy, and NK-based therapy in pancreatic cancer

Positive-selection and ligation-independent cloning vectors for large scale in Planta expression for plant functional genomics

Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligationindependent cloning (LIC) vectors derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC, and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes, alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3' to 5' exonuclease activity in the presence of only one dNTP #dGTP for the inserts and dCTP for the vector#. To make recombinants, the vector plasmid and the insert PCR fragment were annealed at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100% efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance #R# genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic studies in plants.This work was financially supported by grants from the Crop Functional Genomics Center and the National Research Foundation (Project No. 2010-0015105) of Ministry of Education, Science and Technology (MEST) of the Korean Government.OAIID:oai:osos.snu.ac.kr:snu2010-01/102/0000005113/9SEQ:9PERF_CD:SNU2010-01EVAL_ITEM_CD:102USER_ID:0000005113ADJUST_YN:YEMP_ID:A077085DEPT_CD:517CITE_RATE:2.046FILENAME:첨부된 내역이 없습니다.DEPT_NM:식물생산과학부EMAIL:[email protected]_YN:YCONFIRM:

Multiple recognition of RXLR effectors is associated with nonhost resistance of pepper against Phytophthora infestans

Summary Nonhost resistance (NHR) is a plant immune response to resist most pathogens. The molecular basis of NHR is poorly understood, but recognition of pathogen effectors by immune receptors, a response known as effector-triggered immunity, has been proposed as a component of NHR. We performed transient expression of 54 Phytophthora infestans RXLR effectors in pepper (Capsicum annuum) accessions. We used optimized heterologous expression methods and analyzed the inheritance of effector-induced cell death in an F 2 population derived from a cross between two pepper accessions. Pepper showed a localized cell death response upon inoculation with P. infestans, suggesting that recognition of effectors may contribute to NHR in this system. Pepper accessions recognized as many as 36 effectors. Among the effectors, PexRD8 and Avrblb2 induced cell death in a broad range of pepper accessions. Segregation of effector-induced cell death in an F 2 population derived from a cross between two pepper accessions fit 15 : 1, 9 : 7 or 3 : 1 ratios, depending on the effector. Our genetic data suggest that a single or two independent/complementary dominant genes are involved in the recognition of RXLR effectors. Multiple loci recognizing a series of effectors may underpin NHR of pepper to P. infestans and confer resistance durability

Multiple recognition of RXLR effectors is associated with nonhost resistance of pepper against Phytophthora infestans

Summary: Nonhost resistance (NHR) is a plant immune response to resist most pathogens. The molecular basis of NHR is poorly understood, but recognition of pathogen effectors by immune receptors, a response known as effector-triggered immunity, has been proposed as a component of NHR. We performed transient expression of 54 Phytophthora infestansRXLR effectors in pepper (Capsicum annuum) accessions. We used optimized heterologous expression methods and analyzed the inheritance of effector-induced cell death in an F2 population derived from a cross between two pepper accessions. Pepper showed a localized cell death response upon inoculation with P. infestans, suggesting that recognition of effectors may contribute to NHR in this system. Pepper accessions recognized as many as 36 effectors. Among the effectors, PexRD8 and Avrblb2 induced cell death in a broad range of pepper accessions. Segregation of effector-induced cell death in an F2 population derived from a cross between two pepper accessions fit 15 : 1, 9 : 7 or 3 : 1 ratios, depending on the effector. Our genetic data suggest that a single or two independent/complementary dominant genes are involved in the recognition of RXLR effectors. Multiple loci recognizing a series of effectors may underpin NHR of pepper to P. infestans and confer resistance durability

PepMoV cistrons used in this study.

<p>PepMoV cistrons used in this study.</p

Identification of NIb as the HR-inducing avirulence factor against <i>Pvr4</i>-bearing pepper plants.

<p>(A) Transient expression of PepMoV viral proteins in CM334 and Jupiter. Eleven cistrons from PepMoV were infiltrated into CM334 and Jupiter. At 3 dpi, leaves were cleared with 100% ethanol to remove chlorophylls in order to visualize the cell death. For this and subsequent experiments, Empty vector and vector with necrosis-inducing protein (NIP) from <i>P</i>. <i>sojae</i> were infiltrated as a negative or positive control, respectively. Regions of infiltration were marked with ovals and the area of cell death was marked as red. Inoculated viral cistrons were depicted under panels. (B) Transient expression of HC-Pro:HA, CP:HA and NIb:HA in CM334. Plant responses with HA-tagged proteins were tested in <i>Pvr4</i>-harboring plants (CM334). Inoculated viral cistrons were depicted under panels. (C) Expression of PepMoV NIb:HA, CP:HA and HC-Pro:HA proteins in <i>N</i>. <i>benthamiana</i> leaves. 5-week-old tobacco leaves were collected at 24hpi and 48hpi. Untreated leaves were used as mock for negative controls. Each protein was immunodetected by using anti-HA antibody. Coomassie blue–stained total proteins were shown as loading controls.</p

RNA-Dependent RNA Polymerase (NIb) of the Potyviruses Is an Avirulence Factor for the Broad-Spectrum Resistance Gene <i>Pvr4</i> in <i>Capsicum annuum</i> cv. CM334

<div><p>Potyviruses are one of the most destructive viral pathogens of Solanaceae plants. In <i>Capsicum annuum</i> landrace CM334, a broad-spectrum gene, <i>Pvr4</i> is known to be involved in resistance against multiple potyviruses, including <i>Pepper mottle virus</i> (PepMoV), <i>Pepper severe mosaic virus</i> (PepSMV), and <i>Potato virus Y</i> (PVY). However, a potyvirus avirulence factor against Pvr4 has not been identified. To identify the avirulence factor corresponding to Pvr4 in potyviruses, we performed <i>Agrobacterium</i>-mediated transient expressions of potyvirus protein coding regions in potyvirus-resistant (<i>Pvr4</i>) and -susceptible (<i>pvr4</i>) pepper plants. Hypersensitive response (HR) was observed only when a RNA-dependent RNA polymerase (NIb) of PepMoV, PepSMV, or PVY was expressed in <i>Pvr4</i>-bearing pepper leaves in a genotype-specific manner. In contrast, HR was not observed when the NIb of <i>Tobacco etch virus</i> (TEV), a virulent potyvirus, was expressed in <i>Pvr4</i>-bearing pepper leaves. Our results clearly demonstrate that NIbs of PepMoV, PepSMV, and PVY serve as avirulence factors for Pvr4 in pepper plants.</p></div

Correlation of genotypes and cell death phenotype of <i>Pvr4</i> against NIb in the F2 population.

<p>(A) Identification of genotype in relation to <i>Pvr4</i> using the CAPS marker (PCAPS15). Thirty plants of the F2 generation were tested to identify their genotypes. Genotypes of plants (Gen*) are described under the images as R (resistant) or S (susceptible). (B) Response of the F2 population plants derived from Jupiter and CM334 to PepMoV proteins, NIb and CP. Thirty progenies of the F2 generation were tested to verify whether <i>Pvr4</i>-harboring plants show HR in response to PepMoV NIb. The F2 lines which showed HR cell death as well as <i>Pvr4</i> genotypes were marked as R. S represents the F2 lines which did not show HR cell death and were confirmed as <i>pvr4</i>-plants. Inoculated viral cistrons were depicted at the left of panel.</p